Methods have been described for the direct sequencing of genomic DNA which are based on polymerase chain reaction (PCR) [Wong, et al. (1987) and Engelke, et al. (1988)]. Genomic amplification with transcript sequencing (GAWTS), incorporates a phage promoter sequence into at least one of the PCR primers and is described in the parent application Ser. No. 149,312, filed Jan. 28, 1988.
In contrast to autosomal recessive mutations, deleterious X-linked mutations are eliminated within a few generations because the affected males reproduce sparingly if at all. Thus, each family in an X-linked disease such as hemophilia B represents an independent mutation. From the perspective of efforts to understand the expression, processing, and function of factor IX, this is useful since a large number of mutations are potentially available for analysis. In addition to facilitating structure-function correlations, the rapidity of GAWTS makes it practical to perform direct carrier testing and prenatal diagnosis of at risk individuals. By amplifying and sequencing 11 regions of the hemophilic factor IX gene which total 2.8 kb, it should be possible to delineate the causative mutation in the overwhelming majority of individuals as these regions contain the putative promoter, the 5' untranslated region, the amino acid coding sequences, the terminal portion of the 3' untranslated region, and the intron-exon boundaries. Once the mutation is delineated, GAWTS can be used to directly test an at-risk individual, thereby finessing the multiple problems associated with indirect linkage analysis.
Another aspect of the subject invention concerns a direct method for rapidly obtaining novel sequences from clones involving promoter ligation and transcript sequencing, and uses thereof.
The hallmark of the steroid/thyroxine/retinoic acid receptor gene superfamily is a pair of zinc binding "fingers" which determine the specificity of DNA binding [Evans, R. M., Science, 240:889-895(1988). Certain amino acids in the zinc finger DNA binding domains are highly conserved, and recent members of this gene family have been found in Drosophila by analyzing sequences that cross-hydridize in a low stringency Southern blot with a human retinoic acid receptor cDNA probe [Oro, A. E., E. S. Ong, J. S. Margolis, J. W. Posakony, M. McKeown, and R. M. Evans, Nature 336:493-496 (1988)]. The inventor has used the same approach to isolate members of the superfamily in fungi since steroid-specific, high affinity binding proteins have been described in the cytosols of Saccharomyces cervisiae, Paracoccidioides brasiliensis, and Candida albicans [Burshell, A., P. A. Stathis, Y. Do, S. C. Miller, and D. Feldman, J. Biol. Chem, 259:3450-3456 (1984); Feldman, D., Y. Do, A. Burshell, P. Stathis, and D. S. Loose, Science, 218:297-298 (1982); Loose, D. S., and D. Feldman, J. Biol. Chem, 257:4925-4930 (1982); and Loose, D. S., D. J. Schurman and D. Feldman, Nature, 293:477-479 (1981)]. In the water mold Achlya ambisexualis, the receptor for antheridiol (a steroid that regulates sexual physiology) was found to have many of the same properties of steroid receptors in higher eucaryotes [Reihl, R. M., D. O. Toft, M. D. Meyer, G. L. Carlson and T. C. McMorris, Exp. Cell Res. 153:544-549 (1984); and Reihl, R. M., D. O. Toft, J. Biol. Chem., 259:15324-15330 (1984)]
Since false positive signals commonly occur with low stringency Southern blots, the inventor has developed a method called promoter ligation and transcript sequencing (PLATS) to allow rapid analysis of cross-hybridizing segments by reducing the effort required to determine the precise sequence of the segment. In a broader sense, PLATS is a general method for obtaining novel sequence which eliminates lambda DNA purification and subcloning steps which are required by conventional methods. PLATS is illustrated by sequencing a 1.1 kb segment of Achlya ambisexualis which cross-hybridizes to the DNA binding domain of the Xenopus and chicken estrogen receptor. This segment contains a transcribed open reading frame which is not a member of the steriod/thyroxine/retinoic acid receptor superfamily. However, the inventor speculates that the Achlya gene product may belong to a novel class of transcriptional regulators that bind DNA with a zinc finger containing three cysteines and one histidine.
The ability to screen populations for carriers of genetic disease in an accurate, inexpensive, and rapid manner would provide the opportunity for widespread genetic counseling and, ultimately, the possible elimination of such diseases. A successful example of protein based carrier screening is Tay-Sachs disease (G.sub.M2 gangliosidosis type B), which is caused by a deficiency in .beta.-hexosaminidase activity. Since non-carrier and carrier levels of enzymatic activity do not overlap, genetic status can be unequivocally assigned. [Ben-Yoseph, U., J. E. Reid, B. Shapiro, H. L. Nadler., Am. J. Hum. Genet., 37:733-748 (1985)] Screening for Tay-Sachs has reduced markedly the incidence of this disease in Ashkenazi Jews. [O'Brien, J. S., the gangliosidases, In: Stanbury J. B., J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, M. S. Brown, eds. Metabolic Basis of Inherited Disease. New York: McGraw-Hill, 1983:945-969]. Unfortunately, measurements of protein or metabolite levels for other genetic diseases are not usually accurate enough for this type of population screening. Population screening may eventually be possible, however, with DNA-based methods.
Phenylketonuria (PKU) is one disease amenable to DNA-based screening. Classical PKU is an autosomal recessive disease affecting one in 10,000 newborn Caucasians of northern European descent. The disease is the result of a deficiency in hepatic phenylalanine hydroxylase activity (PAH), which causes a primary elevation of serum phenylalanine and secondary abnormalities in compounds derived from aromatic amino acids. [Blau, K. In: Yondim MBH, ed. Aromatic Amino Hydoxylases and Mental Diseases. New York: Wiley, 1979:79-139] If left untreated in infancy, severe mental retardation ensures. While treatment with a low phenylalanine diet can prevent mental retardation, the disease has not been rendered benign. Phenylketonurics still encounter problems, including: 1) failure to reach full intellectual potential due to incomplete compliance with the very stringent dietary therapy [Holtzman, N. A., R. A. Kronmal, W. Van Doorninck, C. Azen, R. Koch, New Engl. J. Med., 314:593-598 (1986)]; 2) a high frequency of birth defects in children of affected females [Scriver, C. R., C. L. Clow, Ann Rev. Genet., 14:179-202 (1980)); and 3) a high incidence of behavioral problems. [Holtzman, et al., (1986); Realmuto, G. M., B. D. Garfinkel, M. Tuckman, M. Y. Tsai, P-N. Chang, R. O. Fisch, S. Shapiro., J. Nerv. Mental Dis., 174: 536-540 (1986)]
Subsequent to the cloning of PAH cDNA, [Kwok, S. C. M., F. D. Ledley, A. G. DiLella, K. J. H. Robson, S. L. C. Woo. Biochem., 24:556-561 (1985)] it was found that 90% of the PKU alleles in the Danish population are confined to four haplotypes. [Chakraborty, R., A. S. Lidsky, S. P. Daiger, F., Guttler, S. Sullivan, A. G. DiLella, S. L. C. Woo., Hum. Genet., 76: 40-46. (1987)] The mutations in haplotypes 2 and 3 represent 20% and 40% of the PKU alleles, respectively. The mutation in haplotype 2 is a C to T transition at amino acid 408 in exon 12 of the PAH gene [DiLella, A. G., J. Marvit, K. Brayton, S. L. C. Woo., Nature, 327:333-336. (1987)] and the mutation in haplotype 3 is a G--A transition at the intron 12 donor splice junction. [DiLella, A. G., J. Marvit, A. S. Lidsky, F. Guttler, S. L. C. Woo., Nature, 322:799-803 (1986)] The mutant alleles associated with haplotypes 2 and 3 are also prevalent in the United States population. [Moore, S. D., W. M. Huang, R. Koch, S. Snyderman, S. L. C. Woo., Am. J. Hum. Genet., 43:A90 (1988)] When the mutations in haplotypes 1 and 4 are defined, 90% of all PKU carriers of northern European descent (approximately 4 million individuals in the United States alone) could be directly diagnosed by DNA methods. The current methods which can detect such point mutations include: i) direct DNA sequencing, [Gyllensten, U. B., H. A. Erlich., Proc. Natl. Acad. Sci., 85:7652-7656 (1988)]; ii) denaturing gradient gel electrophoresis [Myers, R. M., N. Lumelsky, L. S. Lerman, T. Maniatas, Nature, 313:495-498 (1985)], iii) polymerase chain reaction (PCR) followed by allele specific oligonucleotide hybridization [DiLella, A. G., W-M. Huang, S.L.C. Woo., Lancet, 1:497-499 (1988)]; iv) allele specific DNA ligation [Landegren, U., R. Kaiser, J. Sanders, L. Hood, Science, 241:1077-1080 (1988)]; and v) ribonuclease cleavage of mismatched heteroduplexes. [R. M., Myers, Z. Larin, T. maniatas Science, 230:1242-1246 (1985)] However, these techniques in their present form are unlikely to find widespread application in population screening because they lack the requisite speed, technical ease, and/or cost effectiveness.
This invention extends GAWTS by providing a method for rapid and direct access to an mRNA sequence or its protein product which is not limited by either tissue or species specificity. In addition, this application provides a direct method for rapidly obtaining novel sequences from clones involving promoter ligation and trabscript sequencing.
Lastly, the subject invention provides a method for polymerase chain reaction amplification of specific alleles to reliably distinguish between alleles differing in only part.